Effect of hormones and growth factors on the proliferation of adult cricket neural progenitor cells in vitro

In the adult cricket brain, a cluster of neuroblasts produces new interneurons that integrate into the mushroom body (MB), the main associative structure for multisensory information of the insect brain. In previous study we showed the antagonist role of the two morphogenetic hormones, juvenile hormone (JH) and ecdysone, on the regulation of adult MB neurogenesis in vivo. In order to examine whether these hormones act directly on neural progenitor cells, we developed an organotypic culture of MB cortices. Cell proliferation was assessed by 5-bromo, 2'-deoxyuridine (BrdU) incorporation. We showed that JH increased mushroom body neuroblast (MBNb) proliferation, confirming the mitogenic effect of JH observed in vivo. By contrast, ecdysone did not affect the amount of BrdU-labeled nuclei, suggesting that the inhibitory effect observed in vivo probably proceeded from an indirect pathway. We then examined the role of growth factors known to stimulate neural stem cell/progenitor cell proliferation in vertebrates. As shown by calcium imaging, MBNb only expressed functional receptors for insulin whereas mature interneurons responded to IGF-I and bFGF. Both insulin (10 microg/ml) and IGF-I (10 ng/ml) enhanced MB progenitor cell proliferation in culture, although the insulin effect was more pronounced. This effect was abolished when an inhibitor of polyamine biosynthesis was present in the medium, suggesting a link between polyamines and the insulin signaling pathway. By contrast, bFGF (20-200 ng/ml) failed to stimulate MBNb proliferation. Our results point to conserved and divergent mechanisms between vertebrates and invertebrates in the regulation of adult neural progenitor cell proliferation.     

T cell activation in vivo targets diacylglycerol kinase alpha to the membrane: a novel mechanism for Ras attenuation

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid, leading to decreased and increased levels, respectively, of these two lipid messengers that play a central role in T cell activation. Nine DGK isoforms, grouped into five subtypes, are found in higher organisms; all contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. In this study, we have researched in vivo the regulation of DGK alpha, using a transgenic mouse model in which injection of an antigenic peptide activates the majority of peripheral T cells. We demonstrate that DGK alpha, highly expressed in resting T lymphocytes, is subject to complex control at the mRNA and protein levels during in vivo T cell activation. Subcellular fractionation of T lymphocytes shortly after in vivo engagement of the TCR shows rapid translocation of cytosolic DGK alpha to the membrane fraction. At early time points, DGK alpha translocation to the membrane correlates with rapid translocation of Ras guanyl nucleotide-releasing protein (RasGRP), a nucleotide exchange activator for Ras that associates to the membrane through a diacylglycerol-binding domain. To demonstrate a causal relationship between DGK alpha activity and RasGRP relocation to the membrane, we determined RasGRP translocation kinetics in a T cell line transiently transfected with constitutive active and dominant-negative DGK alpha mutants. We show that membrane localization of DGK alpha is associated with a negative regulatory signal for Ras activation by reversing RasGRP translocation. This study is the first demonstration of in vivo regulation of DGK alpha, and provides new insight into the functional role of a member of this family of lipid kinases in the regulation of the immune response.     

Overexpression of Toll-like receptor 4 amplifies the host response to lipopolysaccharide and provides a survival advantage in transgenic mice

Toll-like receptors are transmembrane proteins that are involved in the innate immune recognition of microbial constituents. Among them, Toll-like receptor 4 (Tlr4) is a crucial signal transducer for LPS, the major component of Gram-negative bacteria outer cell membrane. The contribution of Tlr4 to the host response to LPS and to infection with virulent Salmonella typhimurium was studied in four transgenic (Tg) strains including three overexpressing Tlr4. There was a good correlation between the level of Tlr4 mRNA expression and the sensitivity to LPS both in vitro and in vivo: Tg mice possessing the highest number of Tlr4 copies respond the most to LPS. Overexpression of Tlr4 by itself appears to have a survival advantage in Tg mice early during infection: animals possessing more than two copies of the gene survived longer and in a greater percentage to Salmonella infection. The beneficial effect of Tlr4 overexpression is greatly enhanced when the mice present a wild-type allele at natural resistance-associated macrophage protein 1, another critical innate immune gene involved in resistance to infection with SALMONELLA: Tlr4 and natural resistance-associated macrophage protein 1 exhibit functional epistatic interaction to improve the capacity of the host to control bacterial replication. However, this early improvement in disease resistance is not conducted later during infection, because mice overexpressing Tlr4 developed an excessive inflammatory response detrimental to the host.     

Detection of new transposable element families in Drosophila melanogaster and Anopheles gambiae genomes

The techniques that are usually used to detect transposable elements (TEs) in nucleic acid sequences rely on sequence similarity with previously characterized elements. However, these methods are likely to miss many elements in various organisms. We tested two strategies for the detection of unknown elements. The first, which we call "TBLASTX strategy," searches for TE sequences by comparing the six-frame translations of the nucleic acid sequences of known TEs with the genomic sequence of interest. The second, "repeat-based strategy," searches genomic sequences for long repeats and clusters them in groups of similar sequences. TE copies from a given family are expected to cluster together. We tested the Drosophila melanogaster genomic sequence and the recently sequenced Anopheles gambiae genome in which most TEs remain unknown. We showed that the "TBLASTX strategy" is very efficient as it detected at least 332 new TE families in D. melanogaster and 400 in A. gambiae. This was unexpected in Drosophila as TEs of this organism have been extensively studied. The "repeat-based strategy" appeared to be very inefficient because of two problems: (i) TE copies are heavily deleted and few copies share homologous regions, and (ii) segmental duplications are frequent and it is not easy to distinguish them from TE copies.     

Long-lasting occupancy of central nicotinic acetylcholine receptors after smoking: a PET study in monkeys

The aim of this study was to compare the degree of occupancy of central nicotinic acetylcholine receptors (nAChR) in isoflurane anaesthetized baboon brain following inhalation of tobacco smoke (one cigarette containing 0.9 mg nicotine) or i.v. nicotine (0.6 mg i.v.). [18F]Fluoro-A-85380 and positron emission tomography (PET) were used to assess the distribution volumes (DV) of the radiotracer in selected brain areas using a one-compartment model. Eighty minutes after nicotine i.v., DV was reduced by 50 and 66% in the thalamus and putamen, respectively. Six hours after nicotine, a reduction in DV (27% in the thalamus) was still observed. Eighty minutes after inhalation of tobacco smoke, DV was decreased by 52 and 65% in the thalamus and putamen, respectively. Previous PET experiments have demonstrated a short-lasting interaction of [11C]nicotine with nAChRs. Thus, we hypothesized that a metabolite of nicotine with high affinity and long half-live (several hours) could bind at nAChRs. Eighty minutes after a high dose of nornicotine (0.5 mg i.v.), DV was reduced by 53 and 31% in thalamus and putamen, respectively. No significant effect was observed following 0.15 mg nornicotine. Therefore, nornicotine could contribute to the long-lasting occupancy of central nAChRs after smoking.     

Dysfunction of mitochondrial complex I and the proteasome: interactions between two biochemical deficits in a cellular model of Parkinson's disease

Two biochemical deficits have been described in the substantia nigra in Parkinson's disease, decreased activity of mitochondrial complex I and reduced proteasomal activity. We analysed interactions between these deficits in primary mesencephalic cultures. Proteasome inhibitors (epoxomicin, MG132) exacerbated the toxicity of complex I inhibitors [rotenone, 1-methyl-4-phenylpyridinium (MPP+)] and of the toxic dopamine analogue 6-hydroxydopamine, but not of inhibitors of mitochondrial complex II-V or excitotoxins [N-methyl-d-aspartate (NMDA), kainate]. Rotenone and MPP+ increased free radicals and reduced proteasomal activity via adenosine triphosphate (ATP) depletion. 6-hydroxydopamine also increased free radicals, but did not affect ATP levels and increased proteasomal activity, presumably in response to oxidative damage. Proteasome inhibition potentiated the toxicity of rotenone, MPP+ and 6-hydroxydopamine at concentrations at which they increased free radical levels >/= 40% above baseline, exceeding the cellular capacity to detoxify oxidized proteins reduced by proteasome inhibition, and also exacerbated ATP depletion caused by complex I inhibition. Consistently, both free radical scavenging and stimulation of ATP production by glucose supplementation protected against the synergistic toxicity. In summary, proteasome inhibition increases neuronal vulnerability to normally subtoxic levels of free radicals and amplifies energy depletion following complex I inhibition.     

Peptidase activities of the 20/26S proteasome and a novel protease in human brain

Many neurodegenerative diseases are characterized by ubiquitin-positive protein aggregates or inclusion bodies. Ubiquitin-conjugated proteins are degraded by the 20/26S proteasome, and reduced proteasome peptidase activities in brain homogenates have been reported in pathologic lesions of Parkinson's and Alzheimer's diseases. However, it is unknown whether crude extracts of human brain contain other proteases having peptidase activities. We found a novel protease of molecular weight of approximately 105 kDa in normal human brain, which exhibited trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities (corresponding to 52% and 21% of the total activities in crude extracts) but not peptidyl glutamyl peptide hydrolase activity. Both T-L and ChT-L activities of this protease were partially inhibited by proteasome inhibitors (MG132, lactacystin) and, in contrast to those of the proteasome, also by sodium dodecyl sulfate. A simple method to obtain a brain fraction specific to the 20/26S proteasome was developed. Our human brain data suggest that T-L and ChT-L activity levels of the proteasome reported previously may include those of the 105 kDa protease, an enzyme of as yet unknown biological significance, and that it is necessary to separate the proteasome from this protease to evaluate the actual status of the ubiquitin-proteasome system in neurodegenerative disorders.     

The human polycomb group EED protein interacts with the integrase of human immunodeficiency virus type 1

Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4(+) cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.     

Reduced cell turnover in bovine leukemia virus-infected, persistently lymphocytotic cattle

Although nucleotide analogs like bromodeoxyuridine have been extensively used to estimate cell proliferation in vivo, precise dynamic parameters are scarce essentially because of the lack of adequate mathematical models. Besides recent developments on T cell dynamics, the turnover rates of B lymphocytes are largely unknown particularly in the context of a virally induced pathological disorder. Here, we aim to resolve this issue by determining the rates of cell proliferation and death during the chronic stage of the bovine leukemia virus (BLV) infection, called bovine persistent lymphocytosis (PL). Our methodology is based on direct intravenous injection of bromodeoxyuridine in association with subsequent flow cytometry. By this in vivo approach, we show that the death rate of PL B lymphocytes is significantly reduced (average death rate, 0.057 day(-1) versus 0.156 day(-1) in the asymptomatic controls). Concomitantly, proliferation of the PL cells is also significantly restricted compared to the controls (average proliferation rate, 0.0046 day(-1) versus 0.0085 day(-1)). We conclude that bovine PL is characterized by a decreased cell turnover resulting both from a reduction of cell death and an overall impairment of proliferation. The cell dynamic parameters differ from those measured in sheep, an experimental model for BLV infection. Finally, cells expressing p24 major capsid protein ex vivo were not BrdU positive, suggesting an immune selection against proliferating virus-positive lymphocytes. Based on a comparative leukemia approach, these observations might help to understand cell dynamics during other lymphoproliferative disease such as chronic lymphocytic leukemia or human T-cell lymphotropic virus-induced adult T-cell leukemia in humans.     

Furin processing and proteolytic activation of Semliki Forest virus

The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed "p62," which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.